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Aptamer and detection method for C-reactive protein

a technology of c-reactive protein and aptamer, which is applied in the field of aptamer and a detection method relating to creactive protein, can solve the problems of large variation, difficult preservation, and antibodies used in the detection method, and achieve the effect of high sensitivity

Inactive Publication Date: 2014-01-07
NAT CHENG KUNG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The aptamer-based detection method achieves high sensitivity and stability, allowing for accurate and reliable detection of CRP concentrations down to 0.0125 mg / L, improving the assessment and prevention of cardiovascular diseases.

Problems solved by technology

However, the antibodies used in the detection method have disadvantages such as large variation, easily influenced by environment, difficult preservation, and potential contamination of organisms.
Hence, the clinical application of the detection for CRPs is limited and cannot be widely used for the assessment and prevention of diseases.

Method used

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  • Aptamer and detection method for C-reactive protein
  • Aptamer and detection method for C-reactive protein
  • Aptamer and detection method for C-reactive protein

Examples

Experimental program
Comparison scheme
Effect test

experiment 1

Initial Screening of Aptamers Having Affinity to CRPs

1. Establishment of Oligonucleotide Library

[0038]An oligonucleotide library includes 440 types of oligonucleotides. These oligonucleotides are synthesized by Medclub Scientific Co. Ltd., Taiwan, and each has a 72-mer nucleotide sequence shown in a SEQ ID NO:6. The 72-mer nucleotide sequence includes a random sequence constituted by 40 nucleotides (represented by n), a 5′-primer region constituted by 16 nucleotides, and a 3′-primer region constituted by 16 nucleotides:

[0039]5′-ggcaggaagacanaca-[n]40-tggtctgtggtgctgt-3′ (SEQ ID NO: 6). Here, n represents a nucleotide selected from adenine (a), thymine (t), cytosine (c), and guanine (g). The 5′-primer region and the 3′-primer region are respectively designed to be nucleotide sequences recognized by Super-Therm Gold DNA polymerase (Bertec Enterprise Co. Ltd., Taiwan) for performing a polymerase chain reaction (PCR).

[0040]Then, a suitable amount of oligonucleotide library is dissolved ...

experiment 2

Cloning of Aptamers Having Affinity to CRPs

[0049]The PCR product (1 μL) obtained from Experiment 1, pGEM®-T Easy vector (1 μL), 2× rapid ligation buffer (5 μL), and T4 DNA ligase (1 μL) are mixed well, and deionized water is added to make a total volume of the reaction to be 10 μL. The reaction is then placed on ice overnight. Afterwards, 50 μL of JM 109 Escherichia coli (E. coli) competent cells is added to the above solution and mixed well. The reaction is then placed on ice for 20 min. Next, the reaction undergoes heat shock for 45-50 sec in a 42° C. water bath and is rapidly transferred on ice for 2 min. Thereafter, 950 μL of SOC medium is added and incubated under 37° C. in a shaker for 1.5 hr at 150 revolution per min (rpm). The incubated product is well spread on an LB plate containing 1 mg / ML ampicillin, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and 80 μg / mL X-Gal, and incubated overnight at 37° C. Next, fifteen ampicillin-resistant colonies are picked from the LB p...

experiment 3

Screening of Aptamers Having Affinity to CRPs

[0050]Firstly, a 50 mg / L CRP solution and a 1% BSA solution are prepared respectively by dissolving human serum CRP and BSA in 0.05 M carbonate buffer. Next, 50 μL of the CRP solution obtained is added to a 0.2 mL PCR eppendorf and placed under 4° C. for coating overnight. The eppendorf with an inside wall coated with the CRPs is removed from 4° C. and washed with 0.01 M PBS for three times. Thereafter, 50 μL of 1% BSA solution is added and reacted under room temperature for 1 hr. Afterwards, the inside wall of the eppendorf is washed three times with 0.01 M PBS, and an eppendorf A with the inside wall coated with the CRPs is obtained for use.

[0051]In addition, 50 μL of the BSA solution obtained is added to a 0.2 mL PCR eppendorf and placed under 4° C. for coating overnight. The eppendorf with an inside wall coated with BSA is washed three times with 0.01 M phosphate buffer, and an eppendorf B with the inside wall coated with the BSA is o...

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Abstract

An aptamer specifically binding to C-reactive protein (CRP) is provided. The aptamer includes a following nucleotide sequence: 5′-angngggngnntgnnt-3′, wherein n is a nucleotide selected from a, t, c and g.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims the priority benefit of Taiwan application serial no. 99120849, filed on Jun. 25, 2010. The entirety of the above-mentioned patent application is hereby incorporated by reference herein and made a part of this specification.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The invention is directed to an aptamer and a detection method. More particularly, the invention is directed to an aptamer and a detection method relating to C-reactive protein.[0004]2. Description of Related Art[0005]C-reactive proteins (CRPs) are proteins synthesized by the liver and present in plasma. Being a member of the pentraxin family, CRPs have pentagon ring structures constituted by five identical subunits that are non-covalently bonded. Here, each of the subunits includes 224 amino acids and has a molecular weight of approximately 25 kilo-Dalton (kDa). CRPs are mainly synthesized by liver cells reactive to cytokines, and t...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07H21/02
CPCG01N33/68C07K14/4737G01N2333/4737
Inventor LEE, GWO-BINSHIESH, SHU-CHUHUANG, CHAO-JUNELIN, HSIN-I
Owner NAT CHENG KUNG UNIV
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